Fig 1.
Zip7 zinc transport is involved in IL-1 β /TNF- α signaling in RPE cells.
A: Impedance was measured in confluent ARPE-19 cells for 72h in media, media + IL-1 β (5 ng/mL)/TNF- α (10ng/mL), media + IL-1 β /TNF- α + NVS-ZP7-4 or media + IL-1 β /TNF- α + NVS-ZP7-6 (inactive analog of NVS-ZP7-4). B: Challenge of human iPS-derived RPE cells by IL-1β /TNF- α in the presence of NVS-ZP7-4 increased transepithelial resistance relative to cells treated with IL-1β /TNF- α alone. Inclusion of NVS-ZP7-6 during challenge with IL-1β /TNF- α resulted in impedance loss.
Fig 2.
Validation of the role of Zip7 in mediating barrier dysfunction by Zip7 knockdown or overexpression.
A: Cells were transfected with various siRNA oligos and after 72h lysates were made and western blot analysis performed as described in the methods section. siRNA oligo siRNA-1 and siRNA-4 both reduced levels of endogenous Zip7 whereas the expression construct caused increased levels of Zip7 protein in the cell lysates. B: Confluent ARPE-19 cells were treated with10 nM of Zip7 siRNA (s1) or control siRNA (c1) in the presence of 5 ng/mL IL-1 β. Zip7 knockdown increased impedance vs cells transfected with control siRNA. Treatment of cells with IL-1 β reduced impedance and Zip7 knockdown reduced the impedance loss indicating Zip7 contributes to barrier loss induced by IL-1 β. C: Confluent ARPE-19 cells were transfected with a Zip7 expression construct or a vector control and after 48h treated with media or 5 ng/mL of IL-1 β. Zip7 overexpression reduced impedance in resting cells and overexpression of Zip7 reduced impedance during challenge with IL-1 β more than the reduction observed after cells transfected with control vector were challenged with IL-1 β.
Fig 3.
Further characterization of the effects of Zip7 inhibition on cellular barrier function: A: Confluent cells in a Transwell were treated with 5 ng/mL IL-1 β and 10 ng/mL TNF α and 10 μM NVS-ZP7-4 for 48h and 40 kDa FITC-dextran was added to the upper chamber and fluorescence was measured in the bottom chamber after 2h.
Challenge of cells with cytokine increased permeability of the monolayer to FITC-dextran that was blocked by NVS-ZP7-4. B: Cells were treated with IL-1 β /TNF- α in the presence of Zip7 inhibitor NVS-ZP7-4 and inactive analog NVS-ZP7-6. After 48h, cell were harvested and a lysate was subjected to SDS-polyacryamide gel electrophoresis and western blot analysis. IL-1 β /TNF- α induced Cox-2 and reduced occludin. Inclusion of active NVS-ZP7-4 completely blocked these effects whereas NVS-ZP7-6 had no effect. C: F-actin was stained with phalloidin.IL-1 β /TNF- α challenge alone caused stress fiber formation whereas NVS-ZP7-4 treatment alone increased cortical F-actin staining. Stress fiber formation caused by IL-1 β /TNF- α was reduced by NVS-ZP7-4. D: IL-6 induction was inhibited by NVS-ZP7-4 in a dose-dependent manner whereas NVS-ZP7-6 had no effect.
Fig 4.
The role of Zn in signaling of IL-1 β /TNF- α or IL-1 β alone was confirmed using Zn ionophores or Zn chelators.
A: ARPE-19 cells were treated with fluozin3 and ER tracker for 30min, then ZnCl2 (5 μM) and sodium pyrithione (10 μM) was added and cells were cultured for two more hours. Images were visualized with IN Cell Analyzer 2200 as described in the Methods section to visualize Zn levels in cells. Zn/Pyr increased Zn levels in the cell cytoplasm (green) and also in the ER (yellow). B: Cotreatment of ARPE-19 cells with Zn/Pyr and NVS-ZP7-4 increased Zn levels in the er as visualized with ZBR3. C: Cells were challenged with IL-1 β /TNF- α overnight in the presence of NVS-ZP7-4 and increasing concentrations of Zn/Pyr and IL-6 levels in the culture media were determined. Increasing Zn/Pyr restored IL-6 production induced by IL-1 β /TNF- α despite Zip7 inhibition with NVS-ZP7-4. D: Cells were treated with combinations of IL-1 β /TNF- α (10 ng/mL / 20 ng/mL) and western blotting analysis performed. Treatment of cells with 10 ng/mL IL-1 β and 20 ng/mL TNF- α induced Cox-2. Treatment of cells with NVS-ZP7-4 alone induced BiP, CHOP and sXBP-1 as well as occludin. Inclusion of NVS-ZP7-4 during cytokine treatment resulted in inhibition of Cox-2 induction and occludin and the er stress markers BiP, CHOP and sXBP-1. Cytokine treatment with NVS-ZP7-4 and Zn pyrithione resulted in a restoration of Cox-2 induction, and prevented induction of er stress markers and occludin. E: Increasing Zn/Pyr restored impedance loss induced by IL-1 β /TNF- α despite Zip7 inhibition with NVS-ZP7-4. The loss of impedance induced by challenge with IL-1 β /TNF- α was prevented by inclusion of NVS-ZP7-4 during the inflammatory challenge. If cytoplasmic Zn levels were restored using Zn/Pyr, impedance loss was induced by IL-1 β /TNF- α even in the presence of NVS-ZP7-4. F: The cell-permeable Zn chelator TPEN prevented overnight IL-6 induction by IL-1 β indicating intracellular Zn is essential for signaling. Cell toxicity was observed at 10 μM TPEN. G: The cell-impermeable Zn chelator DTPA had no effect on overnight IL-6 induction by IL-1 β indicating extracellular sources of Zn are not required for signaling.
Fig 5.
ER stress contributes to barrier protection caused by Zip7 inhibition.
A: Cells were challenged with IL-1 β (10ng/mL)/TNF- α (20 ng/mL) in the presence or absence of NVS-ZP7-4 and NVS-ZP7-6 and tunicamycin was used as a positive control. 48h after challenge, cells were harvested for western blot analysis. NVS-ZP7-4 and tunicamycin both induced ER stress resulting in increased levels of sXBP-1, CHOP, BiP and a form of PERK with reduced gel mobility. B: Cells were challenged with IL-1 β (10ng/mL)/TNF- α (20 ng/mL) in the absence and presence of NVS-ZP7-4 and either 10μM TUDCA (upper) or 3μM salubrinal (lower). Zip7 inhibition with NVS-ZP7-4 prevented impedance loss induced by IL-1 β /TNF- α but inclusion of TUDCA or salubrinal with NVS-ZP7-4 during the challenge reduced impedance.
Fig 6.
Zip7 inhibition prevents impedance loss in primary human retinal endothelial cells induced by IL-1 β, TNF- α α and glycated BSA.
Impedance was measured in confluent primary human retinal endothelial cells overnight in media, media + IL-1 β (5 ng//mL), media + (TNF- α 5 ng/mL), media + AGE-BSA (250 μg/mL) and media + VEGF (50 ng/mL) in the presence and absence of NVS-ZP7-1. The % increase in impedance when compound was present versus challenge alone was calculated at 24h. Zip7 inhibition reduced impedance loss caused by IL-1 β, TNF- α and glycated BSA but not by VEGF.